PROJECT SUMMARY/ABSTRACT Coronaviruses are major causes of disease worldwide, from the common cold to outbreaks of strains causing severe and sometimes lethal respiratory distress, including SARS, MERS, and the current outbreak of COVID- 19 caused by SARS-CoV-2. It has been observed over the last two decades that multiple coronaviruses interact with components of the autophagy pathway to rearrange cellular membranes and generate sites for RNA replication. In this application, we propose to test the interactions of SARS-CoV-2 with the autophagy pathway. We will rapidly assess whether SARS-CoV-2 has similar interactions with the autophagy pathway as other coronaviruses, then extend the field by identifying how these interactions interfere with viral replication, providing novel therapeutic targets. We will carry this strategy out through three Aims: In the first, we will identify requirements from the early autophagy pathway for SARS-CoV-2 replication. Data from coronavirus studies suggest that the autophagy protein LC3 is recruited to generate double membraned vesicles for virus genomic RNA replication. Other data suggest LC3 is utilized through the ER-associated degradation (ERAD) machinery to generate replication vesicles. We will determine which, if either, of these two pathways is used by SARS-CoV-2. In the second aim, we will determine the role of vesicle acidification in SARS-CoV-2 replication. There is strong evidence that acidification of vesicles plays a role in multiple steps of CoV replication, and we will identify the role of vesicle acidification in SARS-CoV-2 replication. In the third aim, we will identify the specific roles of SARS-CoV-2 non-structural proteins in induction of the autophagic machinery. We have identified previous studies of expression of non-structural proteins in coronavirus and will use this to guide our study of SARS-CoV-2 proteins. These Aims complement existing studies of autophagy in RNA viral life cycles by the Jackson Lab while leveraging the existing expertise of the Frieman Lab in studying cell biology of coronavirus infections. By completing these Aims, we will understand whether SARS-CoV-2 interacts with the autophagy pathway in a similar or unique fashion compared to other coronaviruses, and extend the coronavirus field by identifying specific host-virus interactions that may be targeted for COVID-19 therapies.